Project Abstract Antibiotic resistance currently claims 700,000 lives a year. If the current trends continue, antimicrobial-resistant infections are expected to outpace cancer as a cause of death by 2050. Antibiotic resistance is correlated with the overexpression of particular efflux pumps. These efflux pumps shuttle out most classes of antibiotics so that the antibiotics can?t reach their targets. Efflux pumps have also been shown to be a promising target for otherwise antibiotic tolerant biofilms which are responsible for virtually all chronic bacterial infection. My long term goal is to combat antibiotic resistant infection and infections that have formed biofilms by disabling efflux pumps. Disabling efflux pumps would make the antibiotics we already have work like new by stopping them from being removed from the cell and allowing them to reach their targets. Moreover, disabling efflux pumps in biofilms would act as a potent antibiotic which would cause bacteria to die from their own toxins. Prior efforts to disable efflux pumps have focused on inhibiting one of the drug binding sites of the pump; those efforts have yielded compounds that are toxic and overly specific. The objective of this proposal is to design peptides that prevent the oligomerization of the outer membrane component of the efflux pumps. By targeting the oligomerization of the pump?s ?-strands in its ?-barrel, the resulting pharmaceuticals will be less likely to suffer from toxicity and over-specificity than the current alternatives. Our central hypothesis is that we can disrupt assembly of the outer membrane ?-barrel component of efflux pumps by binding their interface strands with ?-stranded peptides similar to the target-strands? native binding partners. Using this hypothesis, we have designed a proof-of-concept peptide that can disable an efflux pump and sensitize bacteria to antibiotics. However, there have never before been proteins designed to bind to outer membrane proteins, and much of the critical information about those protein-protein interactions is unknown. Therefore, in order to effectively apply this protein design methodology to a broad range of pumps and bacteria species we plan to determine the specificity and affinity markers for the outer membrane-bound oligomerization interactions. We then plan to design high-affinity, efflux pump inhibitors that can combat both antibiotic resistant bacteria and bacteria that have formed biofilms. We will measure specificity and affinity of our interactions in vitro through oligomerization and unfolding studies. We will also study affinity and specificity in bacterial cells by assessment of the level of antibiotic potentiation and biofilm remediation. Ultimately, we plan to make our peptides into drugs that will conquer these two major public health crises. This proposal?s innovation is (1) that we are designing a type of protein-protein interaction that has never been designed before and (2) the novel utilization of an outer membrane protein as a potential pharmaceutical target for combating antibiotic resistance and biofilms. Overall, this work is poised to make a significant contribution because it may enable a revival of existing antibiotics against resistant superbugs and provide novel drugs against antibiotic-tolerant biofilms.